Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates

Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron variant has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta, delta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of both delta and omicron variants relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

Comparison of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates From Rooks (Corvus frugilegus) and Contemporary Human-Derived Strains: A One Health Perspective

During winter, a large number of rooks gather and defecate at the park of a university clinic. We investigated the prevalence of extended-spectrum beta-lactamase (ESBL)–producing Escherichia coli in these birds and compared recovered isolates with contemporary human isolates. In 2016, fecal samples were collected from 112 trap-captured rooks and investigated for presence of ESBL producers using eosin methylene blue agar supplemented by 2 mg/L cefotaxime; 2,455 contemporary human fecal samples of patients of the clinics sent for routine culturing were tested similarly. In addition, 42 ESBL-producing E. coli isolates collected during the same period from inpatients were also studied. ESBL genes were sought for by PCR and were characterized by sequencing; E. coli ST131 clones were identified. Epidemiological relatedness was determined by pulsed-field gel electrophoresis and confirmed using whole genome sequencing in selected cases. Thirty-seven (33%) of sampled rooks and 42 (1.7%) of human stools yielded ESBL-producing E coli. Dominant genes were blaCTX–M–55 and blaCTX–M–27 in corvid, blaCTX–M–15 and blaCTX–M–27 in human isolates. ST162 was common among rooks. Two rook-derived E. coli belonged to ST131 C1-M27, which was also predominant (10/42) among human fecal and (15/42) human clinical isolates. Another potential link between rooks and humans was a single ST744 rook isolate grouped with one human fecal and three clinical isolates. Despite possible contact, genotypes shared between rooks and humans were rare. Thus, rooks are important as long-distance vectors and reservoirs of ESBL-producing E. coli rather than direct sources of infections to humans in our setting.

Assessment of Ceftazidime-Avibactam 30/20-μg Disk, Etest versus Broth Microdilution Results When Tested against Enterobacterales Clinical Isolates

Multidrug-resistant Gram-negative bacteria, especially for extended-spectrum β-lactamases-producing and carbapenemase-producing Enterobacterales , are disseminating rapidly around the world. Treatment options for these infections are limited, which prompt the development of novel or combinational therapies to combat the infections caused by multidrug-resistant pathogens.

A case report of Salmonella enterica serovar Corvallis from environmental isolates from Cambodia and clinical isolates in the UK

Salmonella enterica subspecies enterica serovar Corvallis (S. Corvallis) has been identified as a human pathogen and as a food contaminant. Diarrhoeal disease is a common diagnosis in tourists visiting Southeast Asia, often with unknown aetiology. However, numerous public health institutes have identified Salmonella as a common causative agent when consuming contaminated food and water. Genomic data from environmental isolates from a Cambodian informal market were uploaded to the National Center for Biotechnology Information (NCBI) platform, allowing the novel sequences to be compared to global whole-genome sequence archives. The comparison revealed that two human clinical isolates from England and four of the environmental isolates were closely related, with an average single nucleotide polymorphism (SNP) difference of 1 (0–3 SNPs). A maximum-likelihood tree based on core SNPs was generated comparing the 4 isolates recovered from a Cambodian informal market with 239 isolates of S. Corvallis received from routine surveillance of human salmonellosis in England and confirmed the close relationship. In addition, the environmental isolates clustered into a broader phylogenetic group within the S. Corvallis population containing 68 additional human isolates, of which 42 were from patients who reported recent international travel, almost exclusively to Southeast Asia. The environmental isolates of S. Corvallis isolated from an informal market in Cambodia are concerning for public health due to their genetic similarity to isolates (e.g. clinical isolates from the UK) with known human virulence and pathogenicity. This study emphasizes the benefits of global and public data sharing of pathogen genomes.

Modified poly(L-lysine)-based structures as novel antimicrobials for diabetic foot infections, an in-vitro study

Background: Wound infections occur as sequelae to skin trauma and cause significant hospitalizations, morbidity and mortality. Skin traumas arise more frequently in those with diabetes or cardiovascular disease and in these settings, may be chronic with poorer outcomes including lower limb amputation. Treatment of chronic wound infection is challenging due to antibiotic resistance and biofilm formation by bacteria including S. aureus and P. aeruginosa, which are among the most frequent causative pathogens. Managing these challenging infections requires new molecules and modalities. Methods: We evaluated antimicrobial and anti-biofilm activity of star-shaped poly(L-lysine) (PLL) polymers against S. aureus and P. aeruginosa strains and clinical isolates recovered from wounds including diabetic foot wounds (DFW) in a Dublin Hospital in 2019. A star-shaped PLL polypeptide series, specifically G2(8)PLL20, G3(16)PLL10, G4(32)PLL5 with variation in polypeptide chain length and arm-multiplicity, were compared to a linear peptide, PLL160 with equivalent number of lysine residues. Results: All PLLs, including the linear polypeptide, were bactericidal at 1mM against S. aureus 25923 and P. aeruginosa PAO1, with log reduction in colony forming units/ml between 2.7-3.6. PLL160 demonstrated similar killing potency against 20 S. aureus and five P. aeruginosa clinical isolates from DFW, mean log reductions: 3.04 ± 0.16 and 3.96 ± 0.82 respectively after 1 hour incubation. Potent anti-biofilm activity was demonstrated against S. aureus 25923 but for clinical isolates, low to moderate loss of biofilm viability was shown using PLL160 and G3(16)PLL10 at 50 mM (S. aureus) and 200 mM (P. aeruginosa) with high inter-isolate variability. In the star-shaped architecture, antimicrobial activity was retained with incorporation of 5-mer hydrophobic amino-acid modifications to the arms of the polypeptides (series G3(16)PLL20-coPLT5, G3(16)PLL20-coPLI5, G3(16)PLL20-coPLP5). Conclusion: These polypeptides offer structural flexibility for clinical applications and have potential for further development, particularly in the setting of diabetic foot and other chronic wound infections.

A method for increasing electroporation competency of Gram-negative clinical isolates by Polymyxin B nonapeptide

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a 5-fold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by 8-fold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA-protein complexes into clinically important bacteria.

Antifungal Resistance Trends of Candida auris Clinical Isolates, New York-New Jersey, 2016-2020

About 55% of U.S. Candida auris clinical cases were reported from New York and New Jersey from 2016 through 2020. Nearly all New York-New Jersey clinical isolates (99.8%) were fluconazole resistant, and 50% were amphotericin B resistant. Echinocandin resistance increased from 0% to 4% and pan-resistance increased from 0 to <1% for New York C. auris clinical isolates but not for New Jersey, highlighting the regional differences.